The hippocampus is critically involved in the early stages of declarative learning, and its function and capacity are degraded during normal aging that causes age-associated learning impairments. It has been repeatedly demonstrated that a cellular biomarker of this age-associated learning deficit is the enlarged Ca2+-dependent postburst afterhyperpolarization (AHP) that reduces the intrinsic excitability of CA1 pyramidal neurons in aged subjects. Thus, we have hypothesized that restoring intrinsic excitability of aged CA1 neurons to a young-like state by reducing the AHP using genetic manipulations would rescue the age- related learning deficits. Hence we have designed a research program to identify the candidate proteins for genetic manipulation with the use of recombinant adeno-associated viral (AAV) vectors. In the initial 3.5 years of this MERIT award, we have determined that 1) Ca2+ accumulation in the cytosol evoked with trains of action potentials is greatly elevated in aged CA1 neurons and may underlie the enlarged AHP in these neurons; 2) Ca2+ buffer capacity is increased in aged CA1 neurons, potentially as a cellular mechanism to counteract the increased Ca2+ accumulation; 3) CREB activation (an important cellular mechanism for protein synthesis necessary for learning and for AHP reduction) is impaired in hippocampus of aged rats; and 4) L-type Ca2+ channel (LTCC) expression on the surface of CA1 neurons is elevated in aged rats, which provides a molecular mechanism for the reported increased Ca2+ influx through LTCC in aged CA1 neurons. Based on these findings, we have identified Ca2+ binding proteins, CREB, and LTCC as candidates to rescue the age-related deficits by manipulating their function with AAV vectors. We have created AAV vectors targeting CREB and LTCC, and will continue the systematic characterization of their potential as therapeutics for restoring the age-related deficits. The candidate Ca2+ binding protein genes to manipulate will be determined from protein microarray experiments (a new powerful method to screen expression level changes in hundreds of proteins), and confirmed through literature review and further molecular (e.g., western blot) assays. In addition, we will identify the source(s) of the elevated Ca2+ accumulation in aged CA1 neurons using Ca2+ imaging with two-photon laser scanning microscopy; and thus, reveal additional potential therapeutic targets for intervention. Our goals remain unchanged: to confirm that the AHP is the key regulator of intrinsic excitability and that targeted molecular methods to reduce the AHP in CA1 neurons in aged subjects will lead to successful learning. Continued success will indicate that the protein being manipulated is a viable candidate to target as a therapeutic intervention point for age- associated learning impairments. This research program has clear relevance to understanding and treating neurodegenerative diseases such as Alzheimer's Disease, in which aging is the principal risk factor.